Mapping the transcriptome underpinning acute corticosteroid action within the cortical collecting duct

Abstract

We report the transcriptomes associated with acute corticosteroid regulation of ENaC activity in polarised mCCDcl1 collecting duct cells. 9 genes were regulated by aldosterone (ALDO), 0 with corticosterone alone and 151 with corticosterone when 11βHSD2 activity was inhibited. We validated 3 novel ALDO-induced genes: Rasd1, Sult1d1 and Gm43305 in primary cells isolated from a novel collecting duct reporter mouse. Background Corticosteroids regulate distal nephron and collecting duct Na+ reabsorption, contributing to fluid-volume and blood pressure homeostasis. The transcriptional landscape underpinning the acute stimulation of the epithelial sodium channel (ENaC) by physiological concentrations of corticosteroids remains unclear. Methods Transcriptomic profiles underlying corticosteroid-stimulated ENaC activity in polarised mCCDcl1 cells were generated by coupling electrophysiological measurements of amiloride-sensitive currents with RNAseq. Generation of a collecting-duct specific reporter mouse line, mT/mG-Aqp2Cre, enabled isolation of primary collecting duct cells by FACS and ENaC activity was measured in cultured primary cells following acute application of corticosteroids. Expression of target genes was assessed by qRT-PCR in cultured cells or freshly isolated cells following acute elevation of steroid hormones in mT/mG-Aqp2Cre mice. Results Physiological relevance of the mCCDcl1 model was confirmed with aldosterone-specific stimulation of SGK1 and ENaC activity. Corticosterone only modulated these responses at supraphysiological concentrations or when 11βHSD2 was inhibited. When 11βHSD2 protection was intact, corticosterone caused no significant change in transcripts. We identified a small number of aldosterone-induced transcripts associated with stimulated ENaC activity in mCCDcl1 cells and a much larger number with corticosterone in the absence of 11βHSD2 activity. Cells isolated from mT/mG-Aqp2Cre mice were validated as collecting duct-specific and assessment of identified aldosterone-induced genes revealed that Sgk1, Zbtbt16, Sult1d1, Rasd1 and Gm43305 are acutely upregulated by corticosteroids both in vitro and in vivo. Conclusions This study reports the transcriptome of mCCDcl1 collecting duct cells and identifies a small number of aldosterone-induced genes associated with acute stimulation of ENaC, including 3 previously undescribed genes.